Dominant negative mutants of guanylyl cyclase

Guanylyl cyclase is a membrane-bound protein which transduces information: extracellular signals such as hormones bind at the cell surface and stimulate the enzyme to synthesize the second messenger, cyclic guanosine monophosphate, which is released into the cell and initiates various cellular responses. Guanylyl cyclase regulates various important physiologic pathways such as fluid volume and pressure homeostasis, fertilization, and smooth muscle relaxation; however the details of these pathways have not been completely elucidated. For many proteins, dominant negative inhibitors--proteins mutated to inhibit the wild-type form without altering their level of expression--have been useful in dissecting various cellular and biochemical pathways. Given that guanylyl cyclase may depend on oligomerization for activity, it appeared possible to engineer an inactive mutated form which could combine with wild-type guanylyl cyclase and interrupt the receptor's catalytic activity and hormone-induced signalling.; The project goals were, by mutation, to identify and examine the catalytic domain of GC-A and to construct dominant negative inhibitors. The specific aims were: (1) construct a soluble, active fragment of GC-A to study the catalytic core of the enzyme; (2) determine the oligomeric state of the catalytic core, and whether oligomerization is required for activity; (3) select highly conserved residues for conversion to alanine and determine the effect of mutagenesis on catalytic and dimerization activities; and (4) co-express the various mutants with wild-type GC-A to determine if any have a dominant negative effect on catalysis or signal transduction.; The carboxyl 282 residues (HCAT) were expressed as a soluble, active fragment representing the catalytic core of GC-A. The fragment exists as a dimer, and catalytic activity is lost when dimerization is destroyed. Of eight targeted amino acids, only mutation of aspartate 893 resulted in complete loss of catalytic activity without destroying dimerization. Various experiments demonstrated that HCAT and HCATD893A are both dominant negative inhibitors of signal transduction by the ligand ANP; in addition HCATD893A is a dominant inhibitor of cyclase activity. These mutants appear to be useful tools for future studies aimed at dissecting the physiological pathways of GC-A.